Gas biosensors

Abstract

Microbial biosensors that generate gas outputs in hard-to-image materials using exogenous methyl halide transferase (MHT) genes. By varying the promoter that is fused to the MHT gene, biosensors for different triggers can be made.

What is claimed is: 
     
         1 . A method, comprising:
 a) growing a microorganism comprising an exogenous gene encoding a methyl halide transferase (MHT) in a solid matrix or a partially opaque medium, wherein said gene is under the direct or indirect control of a promoter-of-interest;   b) adding a substrate for said MHT to said solid matrix or a partially opaque medium;   c) capturing a gas released by said microorganism; and   d) measuring an amount of said captured gas, said amount being proportional to an activity of said promoter-of-interest.   
     
     
         2 . The method of  claim 1 , wherein said exogenous gene is genomically integrated into said microorganism. 
     
     
         3 . The method of  claim 1 , wherein said exogenous gene encodes a protein having SEQ ID NO. 1. 
     
     
         4 . The method of  claim 1 , wherein said substrate is a chlorine or bromine or iodine ion. 
     
     
         5 . The method of  claim 1 , wherein said exogenous gene is from  Batis maritima.    
     
     
         6 . The method of  claim 1 , wherein said exogenous gene encodes a MHT having a sequence selected from: 
       
         
           
                 
                 
               
                     
                   SEQ ID NO: 1 
                 
                     
                   MSTVANIAPV FTGDCKTIPT PEECATFLYK VVNSGGWEKC 
                 
                     
                   WVEEVIPWDL GVPTPLVLHL VKNNALPNGK GLVPGCGGGY 
                 
                     
                   DVVAMANPER FMVGLDISEN ALKKARETFS TMPNSSCFSF 
                 
                     
                   VKEDVFTWRP EQPFDFIFDY VFFCAIDPKM RPAWGKAMYE 
                 
                     
                   LLKPDGELIT LMYPITNHEG GPPFSVSESE YEKVLVPLGF 
                 
                     
                   KQLSLEDYSD LAVEPRKGKE KLARWKKMNN 
                 
                     
                     
                 
                     
                   SEQ ID NO: 2 
                 
                     
                   MASAIVDVAG GGRQQALDGS NPAVARLRQL IGGGQESSDG 
                 
                     
                   WSRCWEEGVT PWDLGQPTPA VVELVHSGTL PAGDATTVLV 
                 
                     
                   PGCGAGYDVV ALSGPGRFVV GLDICDTAIQ KAKQLSAAAA 
                 
                     
                   AAADGGDGSS SFFAFVADDF FTWEPPEPFH LIFDYTFFCA 
                 
                     
                   LHPSMRPAWA KRMADLLRPD GELITLMYLA EGQEAGPPFN 
                 
                     
                   TTVLDYKEVL NPLGLVITSI EDNEVAVEPR KGMEKIARWK 
                 
                     
                   RMTKSD 
                 
                     
                     
                 
                     
                   SEQ ID NO: 3 
                 
                     
                   MAGPTTEFWQ ERFEKKETGW DRGSPSPQLL AWLASGALRP 
                 
                     
                   CRIAVPGCGS GWEVAELAQR GFDVVGLDYT AAATTRTRAL 
                 
                     
                   CDARGLKAEV LQADVLSYQP EKKFAAIYEQ TCLCAIHPDH 
                 
                     
                   WIDYARQLHQ WLEPQGSLWV LFMQMIRPAA TEEGLIQGPP 
                 
                     
                   YHCDINAMRA LFPQKDWVWP KPPYARVSHP NLSHELALQL 
                 
                     
                   VRR 
                 
                     
                     
                 
                     
                   SEQ ID NO: 4 
                 
                     
                   MENVNQAQFW QQRYEQDSIG WDMGQVSPPL KAYIDQLPEA 
                 
                     
                   AKNQAVLVPG AGNAYEVGYL HEQGFTNVTL VDFAPAPIAA 
                 
                     
                   FAERYPNFPA KHLICADFFE LSPEQYQFDW VLEQTFFCAI 
                 
                     
                   NPSRRDEYVQ QMASLVKPNG KLIGLLFDKD FGRDEPPFGG 
                 
                     
                   TKDEYQQRFA THFDIDIMEP SYNSHPARQG SELFIEMHVK 
                 
                     
                   D 
                 
                     
                     
                 
                     
                   SEQ ID NO: 5: 
                 
                     
                   MAEVQQNSGN SNGENIIPPE DVAKFLPKTV DEGGWEKCWE 
                 
                     
                   DGVTPWDQGR ATPLVVHLVE SSSLPLGRGL VPGCGGGHDV 
                 
                     
                   VAMASPERYV VGLDISESAL EKAAETYGSS PKAKYFTFVK 
                 
                     
                   EDFFTWRPNE LFDLIFDYVV FCAIEPETRP AWAKAMYELL 
                 
                     
                   KPDGELITLM YPITDHDGGP PYKVAVSTYE DVLVPVGFKA 
                 
                     
                   VSIEENPYSI ATRKGKEKLA RWKKIN 
                 
                     
                     
                 
                     
                   SEQ ID NO: 6 
                 
                     
                   MNLSADAWDE RYTNNDIAWD LGEVSSPLKA YFDQLENKEI 
                 
                     
                   KILIPGGGNS HEAAYLFENG FKNIWVVDLS ETAIGNIQKR 
                 
                     
                   IPEFPPSQLI QGDFFNMDDV FDLIIEQTFF CAINPNLRAD 
                 
                     
                   YTTKMHHLLK SKGKLVGVLF NVPLNTNKPP FGGDKSEYLE 
                 
                     
                   YFKPFFIIKK MEACYNSFGN RKGRELFVIL RSK 
                 
                     
                     
                 
                     
                   SEQ ID NO: 7 
                 
                     
                   MSDPTQPAVP DFETRDPNSP AFWDERFERR FTPWDQAGVP 
                 
                     
                   AAFQSFAARH SGAAVLIPGC GSAYEAVWLA GQGNPVRAID 
                 
                     
                   FSPAAVAAAH EQLGAQHAQL VEQADFFTYE PPFTPAWIYE 
                 
                     
                   RAFLCALPLA RRADYAHRMA DLLPGGALLA GFFFLGATPK 
                 
                     
                   GPPFGIERAE LDALLTPYFD LIEDEAVHDS IAVFAGRERW 
                 
                     
                   LTWRRRA 
                 
                     
                   and 
                 
                     
                     
                 
                     
                   SEQ ID NO: 8 
                 
                     
                   MTDQSTLTAA QQSVHNTLAK YPGEKYVDGW AEIWNANPSP 
                 
                     
                   PWDKGAPNPA LEDTLMQRRG TIGNALATDA EGNRYRKKAL 
                 
                     
                   VPGCGRGVDV LLLASFGYDA YGLEYSGAAV QACRQEEKES 
                 
                     
                   TTSAKYPVRD EEGDFFKDDW LEELGLGLNC FDLIYDYTFF 
                 
                     
                   CALSPSMRPD WALRHTQLLA PSPHGNLICL EYPRHKDPSL 
                 
                     
                   PGPPFGLSSE AYMEHLSHPG EQVSYDAQGR CRGDPLREPS 
                 
                     
                   DRGLERVAYW QPARTHEVGK DANGEVQDRV SIWRRR. 
                 
             
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
                
               
            
           
         
       
     
     
         7 . A method of detecting the activity of a promoter-of-interest, comprising adding a microorganism comprising an exogenous gene encoding a methyl halide transferase (MHT) to a sample, wherein said MHT gene is under the direct or indirect control by a promoter-of-interest, adding a halide salt to said sample, incubating said sample until said halide salt is converted to a gaseous methyl halide, and detecting an amount of said methyl halide gas being emitted from said sample, wherein said amount directly correlates to an activity level of said promoter-of-interest. 
     
     
         8 . The method of  claim 7 , wherein said exogenous gene encoding said MHT is integrated into the genome. 
     
     
         9 . The method of  claim 7 , wherein said promoter-of-interest is a metal-sensing promoter. 
     
     
         10 . The method of  claim 7 , wherein said promoter-of-interest is a stress-sensing promoter. 
     
     
         11 . The method of  claim 7 , wherein said promoter-of-interest is a redox-sensing promoter. 
     
     
         12 . The method of  claim 7 , wherein said promoter-of-interest is a estrogen- or androgen-responsive promoter and wherein said microorganism also comprises an exogenous gene for an estrogen receptor or an androgen receptor. 
     
     
         13 . The method of  claim 7 , wherein said promoter-of-interest is an aromatic hydrocarbon responsive promoter. 
     
     
         14 . The method of  claim 13 , wherein said promoter-of-interest is a benzene, toluene, and xylene (BETX) responsive promoter and wherein said microorganism also comprises an exogenous gene for a transcriptional activator XylR. 
     
     
         15 . A microbial biosensor for a ligand, comprising a microorganism having a ligand activated promoter operatively coupled to a gene encoding a methyl halide transferase, such that when said ligand and a halide salt are present, said ligand binds said promoter, activating expression of said methyl halide transferase, which converts said halide salt to a gaseous methyl halide which can then be detected, thus detecting said ligand. 
     
     
         16 . A quorum sensitive microbial biosensor, comprising a microbe having a constitutive promoter operably linked to a gene whose expression produces a ligand that can exit said microbe, said microbe also comprising a ligand-activated promoter operatively coupled to a gene encoding a methyl halide transferase, such that when a quorum of microbes are present to make sufficient ligand, said ligand binds said ligand-activated promoter, activating expression of said methyl halide transferase, which converts a halide ion to a methyl halide which can then be detected, thus detecting said ligand and indicating a quorum of said microbes. 
     
     
         17 . A stress sensitive microbial biosensor, comprising a microorganism having a stress sensitive promoter operably linked to a gene encoding a methyl halide transferase, such that when said microorganism is under stress, said stress sensitive promoter activates expression of said methyl halide transferase, which converts a halide ion to a gaseous methyl halide which can then be detected, thus detecting said stress. 
     
     
         18 . A microbial biosensor for a toxin, comprising a microorganism having a toxin-sensitive promoter operably linked to a gene encoding a methyl halide transferase, such that when toxin is present, said toxin binds said toxin-sensitive promoter, activating expression of said methyl halide transferase, which converts a halide ion to a gaseous methyl halide which can then be detected, thus detecting said toxin. 
     
     
         19 . A microbial biosensor for a toxin, comprising a microorganism having a constitutive promoter operably linked to a gene whose expression produces a toxin sensing protein that produces a ligand, said microorganism also comprising a ligand activated promoter operatively coupled to a gene encoding a methyl halide transferase, such that when enough toxin is present to make sufficient ligand, said ligand binds said promoter, activating expression of said methyl halide transferase, which converts a halide ion to a gaseous methyl halide which can then be detected, thus detecting said toxin.